Clinical Virology Laboratory Department of Laboratory Medicine, … · 2020-02-07 · VIROLOGY TEST SELECTION ORGANIZED BY VIRUS [Note – Test schedule varies with virus. Contact

Updated 01/20

Clinical Virology Laboratory Department of Laboratory Medicine, Yale New Haven Hospital

Director: Marie L. Landry, M.D. (office 203-688-3475) Laboratory Manager: Maureen Owen, M.T., A.S.C.P. (office 688-1102)

Location: 55 Park St. Clinical Laboratory Building 6th floor, PS619. Lab phone number: 203-688-3524 Standard Hours of Operation: Mon-Fri 7:00 a.m.-to 11:30 a.m.; Sat and Sun 8:00 a.m.-4:30 p.m.

During Winter Respiratory Season (December-March), extended weekend hours are in effect. Call Laboratory for schedule.

TABLE OF CONTENTS PAGES I. GUIDELINES FOR SPECIMEN COLLECTION 1-2 A. Test ordering B. Viral antibody studies C. Specimen collection for viral culture: labeling, timing, collection devices, and holding temperature D. Specimen collection instructions for selected specimens II. VIROLOGY TEST SELECTION ORGANIZED BY VIRUS (alphabetical listing): 3-9 Virus suspected, Clinical Syndromes, Test Selection and Special Instructions and Time to Result III. CLINICAL SYNDROMES: Viruses Associated, Specimens to Collect and Test Method of Choice 10-13 IV. INTERPRETATION OF TEST RESULTS 14-17 V. SERVICES OFFERED AT THE VIROLOGY REFERENCE LABORATORY, VA-CT 18 *************************** *************************** ***************************

I. GUIDELINES FOR SPECIMEN COLLECTION

A. Specimen collection NOTE: SPECIMENS THAT ARE NOT PROPERLY LABELLED WILL BE REJECTED Collect specimens for PCR, antigen, or culture early in illness when viral shedding is maximal. If you have questions, about tests to order, call the laboratory and ask for the Laboratory Manager or Director.

B. Viral antibody studies For immune status testing (past infection), a single serum sample for IgG is generally sufficient. PLEASE NOTE: During acute infection, virus is present, but antibody is often negative.

To detect acute infection by antibody response, both acute and convalescent sera are recommended to show an antibody rise or a seroconversion from negative to positive antibody. Exceptions are virus infections whose clinical symptoms are immune-mediated and thus antibody is usually present at onset of clinical symptoms (e.g. EBV, HBV, parvovirus B19). Note: False positive IgM results commonly mislead clinicians. A seroconversion of IgG is more reliable than IgM for diagnosis of acute infection. Reactivation of latent or persistent viruses, or secondary mumps or measles vaccine failures, may or may not be associated with a positive IgM or rise in IgG. Rather, PCR is recommended.

Updated 01/20 *see List of Virology Tests Performed, on website https://medicine.yale.edu/labmed/sections/virology/

2 C. Collection devices and holding temperature Sample Collection device* Holding temperature Comments

Virus isolation, PCR or antigen test: Swabs Use viral transport medium Refrigerate Viral transport medium with swabs can be obtained from hospital storeroom. NP aspirate Use sterile trap Refrigerate Body fluids, Use sterile leakproof containers Refrigerate Do not dilute body fluids or BAL, stool BAL in transport medium. Tissues Place in tubes containing liquid viral Refrigerate Viral transport medium transport media to keep tissue moist can be obtained from hospital storeroom. Blood Collect 2 lavender top tubes Room temperature Sample must be processed (plasma) Collection time required within specified hrs of collection. Viral antibody test: Blood (serum) Collect 1 gold top tube Room temperature

D. Specimen collection instructions for selected specimens Nasopharynx swab Insert swab deep into nasopharynx, past point of resistance, to posterior pharynx. Gently rotate to dislodge respiratory epithelial

cells. Alternatively, rub nasal turbinate. Remove and place in transport medium. For infants and small children NP aspirate may be preferred.

Throat swab Swab posterior pharyngeal wall, not buccal mucosa, tonsils, tongue or palate. Swab thoroughly.

Throat swabs can be used for lab developed PCR tests, especially for enterovirus, but are not acceptable for DFA testing or for rapid influenza PCR ( GeneXpert).

Saliva swab Neonatal CMV: Place sterile swab in baby's mouth under the edge of the tongue until it is completely saturated with saliva. Insert

saliva-saturated swab into VTM tube. Collect immediately before or 90 minutes after breast feeding to avoid contamination. For older patients, for mumps or CMV testing, saliva can be spit into sterile cup.

Buccal swab For mumps, collect saliva by swabbing mid-buccal mucosa by Stensen’s duct to collect saliva from parotid

glands

Lesion swab Clean lesion with sterile saline soaked gauze pad. Unroof vesicles or remove crusts. Firmly swab base and margins of the lesion, obtaining fluid and cells. After sample collection, clean lesion thoroughly with betadine. If culture desired, do not use disinfectant prior to sample collection since infectious virus may be inactivated.

Rectal swab Stool specimen (not rectal swab) is required for enteric pathogens; swab of rectal mucosa can be done for proctitis. BAL CSF, Stool Collect each in sterile cup.


3 II. VIROLOGY TEST SELECTION ORGANIZED BY VIRUS [Note – Test schedule varies with virus. Contact Lab for details.]

Virus suspected Clinical symptoms Specimens Tests* Special instructions and comments

Time to result

Adenovirus types 1-51 Enteric adeno-virus types 40,41

URI, pharyngitis, pneumonia, conjunctivitis, keratoconjunc-tivitis, hepatitis, hemorrhagic cystitis, gastroenteritis, intussusception, genital infections Disseminated (severely compromised host) Gastroenteritis

NP swab or aspirate Throat, eye swabs, urine, BAL, tissue, stool Plasma (lavender) Stool

Respiratory Virus PCR Panel (RVP) Virus isolation, if lower respiratory tract or tissue. Respiratory virus DFA screen (NP swab only) Quantitative PCR PCR

PCR is more sensitive and more rapid. Virus isolation can detect the “unexpected”. DFA detects only <60% of culture and <40% of PCR positives Viral load in plasma can be monitored; absolute quantification varies for different serotypes

1 d (PCR) 1-14 days (culture) 4 hrs DFA 1-3 days 1-3 days

Arboviruses causing neurologic disease: EEE, WEE, St. Louis encephalitis, LaCrosse, Jamestown Canyon, West Nile, POW) See also Dengue, Chikungunya

Encephalitis, aseptic meningitis, paralytic disease, febrile illness in summertime Note: Different arboviruses are found in other parts of the world. Travel history is key.

CSF Serum (red top), acute and convalescent CSF

IgM and IgG antibody in CSF and serum PCR for WNV if patient cannot make antibody

WNV done in-house. All other arbovirus requests sent out. POW done only at CDC. Early samples can be falsely negative. Cross-reactions often give false positives. May need PRNT to confirm specificity. Only WNV and dengue PCRs available commercially. If traveled, special testing at CDC may be necessary; call lab.

1-4 days WNV 4-7 days for others 2-4 wks if sent to CDC

BK virus (Polyomavirus)

Tubulointerstitial nephritis Hemorrhagic cystitis in bone marrow transplants, ureteral stenosis post kidney transplant

Plasma, to monitor renal transplants Urine

PCR, quantitative PCR, quantitative

Plasma levels of >10,000 copies/ml associated with risk of nephropathy Note: Mutations can lead to falsely low or negative results.

1-3 days

Coronavirus OC43, 229E, NL63, HKU1 MERS-CoV

Common cold viruses; pneumonia Pneumonia, ARDS after travel to Arabian Peninsula

NP swab or aspirate Lower respiratory samples, sputum, NP/OP swabs, paired sera

RT-PCR, included in RVP. RT-PCR and serology

Peaks mid-winter Testing at State Lab or CDC. Needs approval. Contact Hospital Epi. Notify lab, then hand carry to lab. Special Pathogen protocol.

< 1 day 1-3 days

Chikungunya Fever, myalgia, arthralgia, rash, edema

Serum Antibody >day 5 of illness PCR for <5 days of illness

Serum sent out to Quest 7 days


4 Virus suspected Clinical symptoms Specimens Tests Special instructions

and comments Time to result

Cytomegalovirus (CMV) Roche Cobas Ampliprep PCR

Fever, leukopenia, mononucleosis, hepatitis, pneumonia, oral, esophageal and gastrointestinal ulcerations, neurologic syndromes, retinitis Encephalitis Congenital CMV

BAL, tissue biopsies; ocular fluid Plasma (lavender tube) Serum, acute and convalescent Serum CSF (1 ml) Saliva or urine collected at birth Amniotic fluid

PCR (lab developed, LDT) Virus isolation Quantitative PCR (plasma viral load) Antibody (IgM and IgG); Seroconversion to docu- ment primary infection Immune status IgG only PCR (LDT) PCR (LDT) CMV rapid culture

Note: PCR of tissue or BAL can detect positives that are not clinically relevant. Use antibody tests to confirm primary infection or to determine immune status; do NOT use to follow seropositive patients. Note: CMV in blood can contam-inate CSF and give positive PCR To diagnose congenital infection samples must be obtained within 2-3 wks of birth. Positives after 3 wks can be perinatal infection.

1-21 days 1-2 days 1-2 days 1-2 days 1-2 days 1-2 days

Dengue Fever, myalgias, rash; hemorrhagic fever

Serum

Antibody >day 5 of illness PCR for <5 days of illness

Serum sent out to ARUP

7 days

Ebola Fever, myalgia, prostration, diarrhea, vomiting, ARDS, multiorgan failure, hemorrhage

Blood PCR See department and YNHH Viral Hemorrhagic Fever Protocol for details. Special Pathogen alert.

1-2 days

Enterovirus Summer rashes, herpangina, hand-foot-mouth disease, myocarditis, pleurodynia Aseptic meningitis, encephalitis, paralytic disease, rhomboencephalitis Neonatal “sepsis”

Throat swab, stool, skin vesicle swab, biopsy tissue CSF (1 mL) Blood (plasma), CSF and urine

PCR (Note: Coxsackie A viruses may not grow in routine cell cultures.) PCR RT-PCR

Collect stool, not rectal swab, for best results Note: Diagnosis by antibody titer is not practical or reliable. Preferred test for CSF; however, parechovirus and cardiovirus are not detected. EV71 may be detected only in stool despite CNS disease. Parechoviruses order separate PCR.

1-14 days 1 -2 days 1- 2 days




Epstein-Barr virus (EBV)

Infectious mononucleosis (I.M.) Also hepatitis, pneumonitis, neurologic syndromes, hemolytic anemia, thrombocytopenia, hemophagocytic syndrome Lymphoproliferative disease (PTLD) Nasopharyngeal carcinoma (NPC)

Serum Serum CSF (CNS lymphoma) plasma; tissue Plasma (lavender tube) or tissue Blood (red top)

Heterophile antibody (monospot) EBV antibody panel: VCA IgG, VCA IgM, and EBNA antibodies PCR Quantitative PCR EBV antibody panel and VCA IgA antibody

Positive in 90% of adults and <50% of children with I.M. Request if heterophile antibody is negative or if unusual clinical presentation Note: EBV in lymphocytes can give positive CSF PCR in absence of EBV-associated disease Tissue in situ hybridization done by Pathology EBV VCA IgA available sent out; Elevated VCA IgA antibody useful in early NPC detection and monitoring for recurrence

<1 day 1-2 days 1-3 days 1-3 days 3-7 days

Hantavirus Pulmonary Syndrome (HPS)

Pneumonia, ARDS in previously healthy individual

Serum Biopsy tissues

Antibody Virus isolation and PCR

Sent to State Health Dept; form available in the virology laboratory must be filled out. Sent to CDC via State Health Dept

3-7 days Weeks to months

Hepatitis A Acute hepatitis, relapsing hepatitis Immune status, for travelers to HAV endemic areas

Serum Antibody (anti-HAV IgM) Antibody (anti-HAV IgG)

Order only in cases of acute infectious hepatitis; false positives> true positives if low risk Specify "immune status"

1 day 1 day

Hepatitis B See test interpretation on page 16

Acute hepatitis, chronic hepatitis, hepatocellular carcinoma, cirrhosis, polyarteritis nodosa

Serum Serum

HBsAg, anti-HBc total and IgM, anti-HBs, HBeAg and anti-HBe PCR

Can be ordered as single tests, as part of acute hepatitis, hepatitis general, or hepatitis B virus panels Quantitation of HBV DNA should be done prior to therapy and to monitor response. PCR may help clarify atypical serology results.

1 day 1-4 days




Hepatitis C See test interpretation on pages 16, 17

Acute hepatitis, chronic hepatitis, hepatocellular carcinoma, cirrhosis, essential mixed cryoglobulinemia, porphyria

Serum

Antibody (anti-HCV) HCV RNA (RT-PCR) (anti-HCV RIBA not available since 2013) HCV genotype (LiPA)

Can be ordered as a single test, as part of acute hepatitis or chronic hepatitis panels HCV RNA test is used to confirm antibody results instead of RIBA. Quantitation of HCV RNA should be done when therapy is initiated, and to monitor response Used to guide therapy

1 day 1-3 days 2-7 days

Hepatitis D (Delta)

Acute hepatitis, chronic hepatitis, fulminant hepatitis, deterioration of chronic HBsAg carrier

Serum Antibody (anti-HDV or anti-Delta) HDV RT-PCR

Patient must be HBsAg positive to be infected with HDV. Sent out. Used to monitor antibody positive patients.

7 days

Hepatitis E Acute hepatitis, cholestasis; 20% mortality in pregnant women; acute and chronic infection post-transplant, and in compromised hosts

Serum Serum, stool

Antibody (anti-HEV IgM, IgG) RT-PCR for definitive diagnosis

Tests on travelers sent to Quest. Tests on transplant patients sent to CDC.

1-4 weeks

Herpes simplex virus (HSV) types 1 and 2

Esophagitis, proctitis, hepatitis, pneumonia, retinitis; corneal ulcer Cold sore, gingivostomatitis, skin lesions, genital lesions Encephalitis, meningitis Neonatal HSV Hepatitis; disseminated HSV Immune status

Biopsy tissue, mucosal swab, BAL, ocular swab or fluid Lesion swab CSF (1 mL) Swabs for neonatal HSV; blood, CSF Plasma Serum

PCR Virus isolation HSV PCR, Direct HSV culture PCR PCR PCR Type-specific IgG antibody

PCR most sensitive. Direct PCR done twice a day. Not for ocular or neonatal swabs. PCR on CSF done once a day Viral load can be done in disseminated HSV or hepatitis; low level HSV viremia can be seen with simple cold sores and does not indicate dissemination Immune status (carrier state); HSV IgM is not useful

1 day 1-7 days <1 day 1-7 days 1 day 1 day 1-2 days




Human immuno-deficiency virus (HIV) type 1 & 2 (4th generation: IgM, IgG, p24 Ag)

No symptoms, mononucleosis, acute retrovirus syndrome, AIDS, failure to thrive To determine viral load, to diagnose acute HIV infection or neonatal infection. To guide antiretroviral therapy.

Serum Plasma (lavender) allows faster result Serum or plasma Whole blood (2 lavender top tubes) Whole blood (2 lavender top tubes)

Antibody (CLIA) Rapid HIV-1/2 antibody (OraQuick) HIV-1,-2 IgG by Geenius Quantitative plasma RNA by RT-PCR (Roche TaqMan) Resistance genotype

Note: HIV tests require notification of patient, but not signed consent. Call lab to facilitate rapid result; done by Core Lab when Virology is closed All positive 4th gen require IgG antibody differentiation. Do PCR if IgG negative to rule out acute HIV. In acute infection, viral load should be high Requires 400-1,000 copies/mL

<1 day 30 min 1 day 1-3 days 1-2 weeks (sent out)

Human herpesvirus type 6 (HHV-6A and B)

Roseola infantum, febrile seizures, infectious mono, hepatitis, pneumonitis, encephalitis in HSCT To confirm primary infection

CSF, plasma Serum

PCR (quantify plasma) Antibody, IgM and IgG

Patients with chromosomally integrated HHV-6 have high levels of HHV-6 DNA in blood and CSF HHV-6 antibody rarely useful since infection is universal by age 2

1-3 days 1-2 wks

Human meta-pneumovirus

URI, pneumonia, bronchiolitis NP aspirate, swab, BAL

RT-PCR, in RVP;

Peaks in late winter, early spring 1 day

HTLV I/II

Tropical spastic paraparesis or HTLV associated myelopathy; human T cell leukemia/lymphoma

Serum Whole blood (2 lavender)

Antibody (EIA screen; all positives need confirma-tion by Western blot) Qualitative PCR on PBMCs; not viral load

EIA does not distinguish HTLV-I from II; (W. blot sent out). Positive serology indicates carrier. PCR useful if antibody tests are indeterminate (sent out)

3-7 days 7-14 days

Influenza A, B Avian influenza (A/ H5N1, H7N9, etc)

Influenza syndrome, URI, bronchitis, bronchiolitis in infants, pneumonia, myoperi-carditis, myositis Pneumonia, ARDS, diarrhea, neurologic disease

Nasopharynx (NP) swab, wash, or aspirate, endotrach-eal aspirate, BAL BAL, endotracheal aspirate, sputum (not NP swab)

Rapid influenza PCR in ED, outpatients Included in RVP A subtype or B lineage by PCR if hospitalized Not subtypeable by seasonal virus PCRs.

Collect specimens first 2-4 days of illness; samples collected in first 24 hrs can be falsely negative Notify lab; requires BSL3 safety precautions; initial tests may be negative; repeat testing needed

1-14 hrs 4-48 hrs




JC virus (Polyomavirus)

Progressive multifocal leukoencephalopathy (PML)

CSF Brain biopsy

PCR Histopathology; EM to detect viral particles

Sensitive NIH assay used. Done by Pathology

1-3 days 4-14 days

Measles Coryza, conjunctivitis, rash, Koplik's spots; giant cell pneumonia or respiratory symptoms without rash in compromised hosts; encephalitis; atypical measles in previously immunized

NP swab, urine Serum, acute and convalescent

PCR Antibody, IgM and IgG IgG avidity testing at CDC

Collect early in disease; notify Hospital Epidemiology and the laboratory prior to sample collection; requires approval from State Epi; done at CT DPH Lab IgM, IgG avidity sent to CDC; IgM false positive and false negative

2-3 days 1-7 days

Mumps Parotitis, orchitis, meningitis, encephalitis

Saliva, urine, CSF, Serum, acute and convalescent

PCR Antibody IgG and IgM IgG avidity testing at CDC

Sent to CT DPH; please notify the laboratory IgM sent to CDC; IgM prone to false positive and false negative

3-14 days 1-7 days

Norovirus Gastroenteritis, common source outbreaks (food, shellfish, contaminated water or ice)

Stool collected within 48 hrs of onset of symptoms

RT-PCR (detects genogroups I and II)

Genetic variation in virus strains can lead to falsely negative results; samples should be collected early in illness for best results

1-3 days

Papillomavirus (over 150 types)

Warts, cervical dysplasia Cervical swab or biopsy

PCR Tests only available for genital HPV Sent to Yale Pathology Lab

2-7 days

Parainfluenza types 1-4

URI, croup, bronchitis, pneumonia

NP swab or aspirate, tracheal aspirate, BAL, lung tissue

PCR (in Resp Virus PCR Panel) DFA for outpatient pediatrics Virus isolation

PCR for types 1-4 DFA for types 1-3 only

1 day 4 hrs 3-10 days

Parechovirus types 1-16

Similar to enterovirus; especially neonatal sepsis

NP, blood, CSF, stool, urine

RT-PCR

Order Parechovirus RT-PCR; not detected by enterovirus PCR

1-2 days 3-14 days

Parvovirus B19 Erythema infectiosum (fifth disease), arthralgias, various exanthems and enanthems, aplastic crisis, chronic anemia in compromised hosts, nonimmune hydrops fetalis

Serum Serum; bone marrow; amniotic fluid

Antibody, IgG and IgM PCR

Immunocompromised hosts may not develop antibody PCR can be positive for months after infection; infection may be persistent, especially in compromised hosts

1-3 days 1-3 days




Rabies Clinical rabies, ascending paralysis, rapidly progressive encephalitis Note: In U.S., half of rabies cases report no history of animal bite. Bats are main source in U.S. cases.

Brain biopsy, skin biopsy from nape of neck (to include hair follicles), corneal scrapings ; saliva Blood (red top) and CSF

Rabies antigen (immunostain of tissue) RT-PCR, culture Antibody

Sent to CDC Rabies Lab (www.cdc.gov/ncidod/dvrd/rabies/Professional/professi.htm) Note: Serum test invalid if rabies immune globulin has been given

2-14 days 7-14 days

Respiratory syncytial virus

Bronchiolitis, pneumonia, URI NP aspirate or NP swab, BAL, lung tissue

RT-PCR (in Resp Virus PCR Panel) DFA outpatient pediatrics Virus isolation

RT-PCR and DFA >> RSV culture 1 day 4 hrs 3-14 days

Rhinovirus URI; lower tract disease in infants, asthma, COPD, ICH

NP swab or aspirate; BAL

RT-PCR (in Resp Virus PCR Panel) Virus isolation

RT-PCR much more sensitive than culture; group C does not grow in culture

1 day 3-14 days

Rotavirus groups A, B

Infantile gastroenteritis (group A); diarrhea in adults (group B)

Stool Rotavirus antigen (ELISA)

ELISA detects only group A and may yield false positive results in neonates

1-3 days

Rubella Rubelliform rash, post-auricular adenopathy, arthralgias, congenital rubella syndrome

Serum, acute and convalescent Tissue, throat swab and urine

Antibody RT-PCR

IgG done in-house, IgM sent out if rubella high risk- notify laboratory Special request; please notify laboratory; sent to CDC

1-7 days 3-14 days

Varicella-zoster virus (VZV)

Chicken pox, herpes zoster, pneumonia, neurologic syndromes, retinal necrosis Pneumonia Meningitis, encephalitis, uveitis Immune status CNS vasculopathy

Skin lesion swabs BAL, tissues CSF (1 ml); ocular fluid Serum CSF

VZV Direct PCR PCR PCR Antibody, IgG

Need vigorous swab of lesion to dislodge cells; PCR most sensitive DFA discontinued July 2015. Note: VZV PCR of CSF can be positive in uncomplicated zoster Collect serum promptly after exposure to determine immune status; IgM is not useful CSF IgG useful if testing delayed and PCR is negative

1 day 1-2 days 1-2 days

West Nile virus (WNV)

See Arboviruses


10 III. CLINICAL SYNDROMES: Most Commonly Associated Viruses, Specimens to Collect and Diagnostic Test of Choice Clinical Syndrome Viruses associated Specimens to collect Test method of choice Respiratory Pneumonia

Influenza A, B Adenovirus Respiratory syncytial virus Human metapneumovirus Parainfluenza Rhinovirus Coronavirus (229E, OC43, NL63, HKU1) Cytomegalovirus Varicella-zoster Herpes simplex Hantavirus* MERS CoV*

NP aspirate or swab, BAL, lung tissue NP aspirate or swab, BAL, lung tissue NP aspirate or swab, BAL, lung tissue NP aspirate or swab, BAL, lung tissue NP aspirate or swab, BAL, lung tissue NP aspirate or swab, BAL, lung tissue NP aspirate or swab, BAL, lung tissue BAL, lung tissue; blood BAL, lung tissue BAL, lung tissue Lung tissue, serum Deep respiratory sample, NP/OP, paired sera

RT-PCR, Culture PCR, Culture RT-PCR, Culture RT-PCR RT-PCR, Culture RT-PCR, Culture RT-PCR PCR; Culture PCR; Culture PCR; Culture Serology, PCR, Culture RT-PCR, Serology

URI/pharyngitis

Rhinovirus, Coronaviruses Respiratory syncytial virus Adenovirus Parainfluenza Influenza A,B Enterovirus, parechovirus EB virus

NP aspirate or swab NP aspirate or swab NP aspirate or swab NP aspirate or swab NP aspirate or swab Throat and/or NP swab Serum

RT-PCR; Culture (RV) RT-PCR, DFA; Culture PCR, DFA, Culture PCR, DFA, Culture RT-PCR; DFA, Culture RT-PCR; Culture Serology

Pleurodynia Enterovirus TS, NP swab RT-PCR Ocular Conjunctivitis/ keratitis/ retinitis

Enterovirus Adenovirus Herpes simplex virus Varicella-zoster virus Cytomegalovirus Vaccinia Measles

Conjunctival/ corneal swab, TS Conjunctival/ corneal swab, NP Conjunctival/ corneal swab; ocular fluid Conjunctival/ corneal swab, ocular fluid Ocular fluid Conjunctival/ corneal swab, lesion swab Conjunctival/ corneal swab, NP, serum

RT-PCR PCR PCR, culture PCR PCR Culture (Special request) RT-PCR, serology

Infectious mononucleosis

EB virus Cytomegalovirus Adenovirus HIV HHV-6

Serum Blood, urine, saliva; serum NP swab, TS, urine Serum; blood Serum

Serology PCR, Culture; serology PCR, Culture Serology; PCR PCR, serology

Cutaneous and mucous membrane Vesicular/ ulcerative

Herpes simplex virus Varicella-zoster virus Enterovirus Vaccinia Cytomegalovirus (ICH) Adenovirus (ICH)

Lesion swab Lesion swab TS, stool, lesion swab Lesion swab Lesion swab; blood Lesion swab; throat, stool

PCR, Culture PCR, Culture RT-PCR Culture PCR, Culture PCR, Culture


11 Clinical Syndrome Viruses associated Specimens to collect Test method of choice Papillomas, papules Papillomavirus

Molluscum contagiosum Biopsy Biopsy

Contact Pathology Dept for options (Note: Clinical diagnosis usually sufficient)

Exanthematous Measles Rubella Enterovirus Parvovirus B19 Human herpesvirus type 6 Dengue West Nile Epstein-Barr virus Adenovirus Cytomegalovirus

NP swab or TS, serum Serum; NP swab or TS, urine, tissue TS, stool Serum Serum Serum Serum, CSF Serum NP swab or TS, urine; stool Blood, urine, saliva

PCR; rapid culture; serology PCR, Serology RT-PCR Serology; PCR PCR; Serology Serology; PCR Serology; PCR Serology PCR, culture, DFA PCR (blood), Culture; serology

Cardiovascular Myocarditis/ Pericarditis

Enterovirus Cytomegalovirus Influenza Adenovirus Rhinovirus group C EBV

TS, stool, endocardial biopsy Blood, urine, endocardial biopsy NP swab, [endocardial biopsy-most negative] NP swab or TS, urine; stool NP swab or TS; pericardial fluid Serum

RT-PCR; Culture PCR, Culture RT-PCR, culture PCR culture, DFA RT-PCR Serology

Digestive tract Gastroenteritis Colitis Proctitis

Rotavirus Norovirus Adenovirus Parechovirus Enterovirus (not common) Cytomegalovirus Herpes simplex virus

Stool Stool Stool Stool Stool GI biopsy, blood Lesion swab, rectal swab

ELISA, PCR RT-PCR PCR RT-PCR PCR PCR PCR, culture

Hepatitis

Hepatitis A Hepatitis B Hepatitis C Hepatitis D Hepatitis E EB virus Cytomegalovirus Adenovirus Herpes simplex virus

Serum Serum Serum Serum Serum Serum Liver tissue, blood Liver tissue Liver tissue, blood

Serology Serology; PCR Serology; RT-PCR Serology, RT-PCR Serology, PCR Serology; PCR PCR, Culture PCR, Culture PCR; Culture Note: PCR blood very high viral load in HSV hepatitis; allows rapid diagnosis


12 Clinical Syndrome Viruses associated Specimens to collect Test method of choice Hematologic Bone marrow suppression

EBV Cytomegalovirus Human herpesvirus type 6 Hepatitis A, B, C Parvovirus B19 Influenza Adenovirus HIV

Serum, bone marrow Blood, bone marrow Serum, bone marrow Serum Serum, bone marrow NP aspirate or swab Throat, stool, blood, bone marrow Serum; plasma

Serology, PCR PCR PCR, serology Serology, PCR Serology, PCR RT-PCR, culture PCR, culture Serology, RT-PCR

Virus associated hemophagocytic syndrome

EBV Cytomegalovirus Varicella-zoster Herpes simplex Adenovirus Human herpesvirus type 6 Parvovirus B19

Serum, bone marrow Blood, bone marrow Skin lesions, bone marrow Skin lesions, bone marrow Throat, stool, bone marrow Serum, bone marrow Serum, bone marrow

Serology, PCR PCR, culture PCR, culture PCR, culture PCR, Culture PCR, serology Serology, PCR

Hemolytic anemia EBV Cytomegalovirus Hepatitis B Measles Mumps Rubella

Serum, bone marrow Blood, bone marrow Serum Serum, throat and urine Serum, throat and urine Serum, throat and urine

Serology, PCR PCR, culture Serology RT-PCR, serology, culture RT-PCR, serology, culture RT-PCR, serology

Atypical lymphocytes EBV Cytomegalovirus Hepatitis A, B, C Measles Mumps Rubella Respiratory syncytial Parvovirus B19 HIV

Serum, bone marrow Blood, bone marrow Serum Serum, throat and urine Serum, throat and urine Serum, throat and urine NP aspirate or swab Serum Serum; plasma

Serology, PCR PCR, culture Serology RT-PCR, serology, culture RT-PCR, serology, culture RT-PCR, serology, culture RT-PCR, DFA, culture Serology, PCR Serology, RT-PCR

Neutrophilia Mumps Hepatitis B Viral hemorrhagic fevers**

Serum, throat and urine Serum Serum (biosafety precautions)**

RT-PCR, serology, culture Serology RT-PCR, serology (BSL 3 or 4)

Aplastic anemia Hepatitis C Serum, bone marrow Serology, PCR Pure red cell aplasia Parvovirus B19

Hepatitis C Serum, bone marrow Serum, bone marrow

Serology, PCR Serology, PCR


13 Clinical Syndrome Viruses associated Specimens to collect Test method of choice Neurologic Encephalitis

Herpes simplex virus type 1>>2, except neonatal HSV type 2> 1 Cytomegalovirus Varicella-zoster EBV Arbovirus (EEE, WEE, SLE, West Nile, POW, etc)* Adenovirus Measles, Rubella Mumps Influenza Enterovirus Parechovirus HIV BKV HHV-6 (beware ciHHV6) Rabies* LCMV (transplant)

CSF; brain biopsy CSF, blood CSF, autopsy tissue CSF, lesion swab Serum, CSF CSF and serum CSF, TS, stool NP swab, urine, serum CSF, urine; serum NP swab or TS, CSF CSF, TS, stool (serum in neonates) CSF, TS, stool (serum in neonates) CSF; serum CSF; urine CSF Brain biopsy; Skin biopsy, Saliva; serum, CSF Serum , CSF

PCR; culture PCR PCR, culture PCR Serology; PCR Serology; RT-PCR PCR, culture RT-PCR, serology; culture RT-PCR, serology; culture RT-PCR, DFA, Culture RT-PCR RT-PCR PCR; serology PCR PCR DFA, (for antigen); PCR PCR, serology, Culture Serology; RT-PCR

Meningitis Enterovirus, parechovirus Herpes simplex virus type 2>> 1 Varicella-zoster EBV HIV (acute infection) Mumps WNV, Jamestown Canyon* Lymphocytic choriomeningitis virus (LCMV)

CSF, stool, TS (CSF, serum in neonates) CSF, lesion swab CSF, lesion swab Serum, CSF Plasma, CSF CSF, urine; serum CSF, Serum Serum, CSF

RT-PCR PCR; Culture PCR Serology; PCR RT-PCR, serology RT-PCR, serology; culture Serology Serology

Progressive multifocal leukoencephalopathy

Polyomavirus (JC) CSF; Brain tissue PCR; histopathology; EM

Abbreviations: Specimens: NP, nasopharyngeal swab or aspirate (provides results superior to TS for respiratory viruses); TS, throat swab; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; ciHHV6= chromosomally integrated HHV-6 in 1% of population. Test Methods: ELISA, enzyme linked immunosorbent assay; DFA, direct fluorescence assay; EM, electron microsopy; PCR, polymerase chain reaction’ RT-PCR, reverse transcriptase polymerase chain reaction Please Note: Acute and convalescent serum should be collected for antibody studies. Serologic testing is not practical for enteroviruses, rhinoviruses, papillomaviruses. and polyomaviruses. * Testing is done at the State Laboratory and/or CDC; call the Virology Laboratory for details and to fill out required forms **Notify Health Department and CDC.


14 IV. INTERPRETATION OF TEST RESULTS

VIRUS ISOLATION Please Note: Comprehensive culture now used only for lower respiratory tract samples. HSV culture for genital samples. Rapid CMV culture for

selected samples. PCR preferred test. Cultures of CSF and stool discontinued July 2015. Viruses isolated in routine cell cultures

Interpretation of positive culture

Adenovirus, cytomegalovirus, enteroviruses, herpes simplex, influenza A and B, parainfluenza types 1-4, rhinoviruses, RSV, vaccinia, varicella-zoster virus

Indicates infectious virus present. Significance varies with virus, specimen source and clinical setting. For example, latent viruses can reactivate with or without symptoms (e.g. CMV, HSV, adenovirus). Isolation of other viruses occurs only with acute infection (e.g. influenza).

NOTE: Respiratory Virus PCR Panel is more sensitive than culture or DFA. On outpatients, when a less expensive test is desired, respiratory

screen DFA can be ordered, but results are best in young children. Performance of antigen tests vs culture and PCR is given below.

VIRAL ANTIGEN [DFA] Virus Sample Test Sensitivity DFA vs.

Culture PCR Interpretation of positive result

Influenza A and B NP swab DFA 85-90% 55-85% Culture and DFA associated with acute infection; PCR remains positive longer RSV NP swab DFA 99% 55-80% Culture and DFA associated with acute infection; PCR remains positive longer Adenovirus NP swab DFA 60% 25% Culture and DFA associated with acute infection; PCR remains positive longer Parainfluenza types 1-3 NP swab DFA >90% 60-70% Culture and DFA associated with acute infection; PCR remains positive longer HMPV, Para-4, Rhinoviruses, Coronaviruses

NP swab Not avail-able

Insensitive For all respiratory viruses, low levels of nucleic acid by PCR can reflect recent past infection, poor sample collection, or possibly primary/probe mismatch

*DFA results are best in young children, who shed high titers of virus.. Specificity at YNHH for DFA and PCR is >99% for all tests. Note: If sample poorly collected or collected late in illness, results of all tests will be poor. NP = nasopharynx DFA= direct fluorescent antibody (immunostain of respiratory epithelial cells) Respiratory Screen DFA includes Influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. Respiratory virus PCR panel includes viruses in DFA screen plus HMPV, rhinovirus, parainfluenza 4, and coronaviruses 229E, OC43, NL63, HKU1.

CLOSTRIDIUM DIFFICILE TEST ALGORITHM

1. Perform Rapid GDH Ag & toxin EIA using C.diff QuikCheck Complete (4 times a day; 30 min test TAT) 2. Both negative: Final Report - C. difficile not detected 3. Both positive: Final Report - Positive toxin in a patient with diarrhea is indication for therapy

4. GDH Ag positive, rapid toxin negative, do cytotoxin neutralization in cell culture (4-48 hr TAT) 5. If GDH positive and cytotoxin negative; Final Report interpretation is colonization 6. If GDH positive and cytotoxin positive: Final Report- A positive cytotoxin n a patient with diarrhea is an indication for therapy.

For more details, see C. difficile newsletter, https://medicine.yale.edu/labmed/sections/virology/newsletter/ Volume 26 (1), Nov. 2017


15 VIRAL ANTIBODY

Please note: Administration of blood products or immunoglobulin may result in passive transfer of antibody and transiently positive antibody test results. False negative antibody results may occur in immunocompromised hosts or agammaglobulinemic patients. IMMUNE STATUS TESTING Sensitivity and specificity of these assays ranges from 97-99% in various studies. Virus Method Result Interpretation Cytomegalovirus, Herpes simplex, Measles, Rubella, Varicella-zoster

CLIA Negative Positive Equivocal

-No antibody detected -Antibody present -Non-specific reaction or low level antibody. Submit second sample.

CLIA= chemiluminescence immunoassay DIAGNOSIS OF ACUTE INFECTION Virus Method IgG Result IgM Result Interpretation Cytomegalovirus CLIA -

+ + -

+ + - -

Primary infection (or false positive) Primary or reactivation infection (Note: CMV IgM rise can be due to EBV, and vice versa) Past infection No antibody detected

Parvovirus EIA + or – + -

+ - -

Acute infection (or false positive) Past infection No antibody detected

West Nile Virus EIA - or + + -

+ - -

Acute infection or false positive Past infection with flavivirus; cross reaction with CMV or enterovirus Uninfected or early in infection; positive IgM make take 8 days

PATTERNS OF EBV-SPECIFIC ANTIBODY RESULTS AT DIFFERENT STAGES OF INFECTION: Antibody to EBV antigens: Uninfected Primary Past Reactivationb Viral capsid antigen (VCA)-IgG - ++ +a ++ Viral capsid antigen (VCA)-IgM - + - + or - Epstein-Barr nuclear antigen (EBNA) - - + + a, High titers to EBV VCA IgG may persist for years after primary infection in healthy individuals. b, To link EBV serologic reactivation with clinical disease requires tissue EBV PCR or hybrdization; high viral load by PCR in blood may also be helpful


16 HEPATITIS TEST RESULTS

Positive Result Interpretation HBsAg

Active hepatitis B infection. Detectable during incubation period, acute hepatitis, and chronic HBV infection. Patient is considered infectious. Persistence beyond 6 months indicates chronic infection.

Anti-HBs

Marker of recovery and immunity. Detectable 1-3 months after HBsAg disappears. Indicates previous hepatitis B, immunization with HBV vaccine, or passive antibody via hepatitis B immune globulin.

Anti-HBc (total antibody)

Detects both IgG and IgM. Indicates current or past hepatitis B infection. Present during the "window" period when HBsAg has disappeared, but anti-HBs is not yet detectable. May persist longer than anti-HBs and be the only marker for past HBV infection. Not associated with recovery or immunity. If anti-HBc is the only positive HBV test, it may indicate past infection, non-specific result, or low-level chronic HBV infection. Perform HBV DNA PCR to exclude chronic HBV infection.

Anti-HBc IgM

Consistent with recent hepatitis B infection. Antibody usually persists 4-6 months after acute stage. Occasionally present in chronic active hepatitis. Test done routinely on all samples positive for anti-HBc but negative for anti-HBs.

HBeAg

Serum contains HBV e antigen. This suggests that the patient is highly infectious. Persistence beyond 10 weeks suggests chronic liver disease.

Anti-HBe

Anti-HBe appears prior to loss of HBsAg and signals reduced level of infectious virus. Suggests early convalescence or past infection with HBV, but may also be seen in HBsAg carrier state.

Anti-Delta

Delta is a defective virus causing hepatitis only in association with HBV. Delta can be acquired simultaneously with HBV (coinfection) or as a superinfection in HBV carriers. Patients with delta virus infection have anti-delta antibody in their serum.

Anti-HAV IgG

Positive result is consistent with current or past hepatitis A, immunization or passive antibody from immune globulin. Patients with anti-HAV IgG are usually immune to further HAV infection and are not infectious.

Anti-HAV IgM Positive test indicates recent infection with HAV. IgM anti-HAV persists for about 4-6 months after acute infection. Low positive results can be non-specific. Test should only be ordered in cases of acute infectious hepatitis.

Anti-HCV

Indicates infection with hepatitis C. Negative results do not exclude infection with HCV, since antibody levels may be below assay detection limits. Detection or quantitation of HCV RNA in serum can be helpful in assessing disease activity.

COMMON HEPATITIS B SEROLOGY PATTERNS

HBsAg Anti-HBc IgM Total anti-HBc Anti-HBs No evidence of HBV infection - - - - Acute HBV infection + + + - Chronic HBV (if HBsAg+ for > 6 months) + - + - "Window" period during acute HBV - + + - Previous HBV infection - - + + HBV vaccine response - - - + Note: HBV DNA PCR may aid in clarifying atypical serologic patterns.


17 HEPATITIS C VIRUS (HCV) ANTIBODY TEST INTERPRETATION Note: RIBA currently not available in U.S. Use PCR for confirmation. CLIA RNA Interpretation

- N.D. A negative CLIA result does not absolutely exclude HCV infection. Antibodies are not detectable for 6-7 weeks after initial infection or may not develop in compromised hosts. In high risk individuals, repeat antibody testing in 2 months and/or HCV RNA PCR should be considered.

+ High

N.D. A high level of antibody was detected in this specimen. If this patient is not known to have HCV infection, a confirmatory HCV RNA PCR should be ordered

+ Low

N.D. A low level of reactivity was detected in this specimen. This may be a false-positive reaction, but may represent a low level of HCV-specific antibody. Depending on the clinical circumstances, HCV PCR to detect active viremia and/or repeat antibody testing in 4-6 weeks is recommended.

+ - Not currently infected. Recovered or false positive screen.

+ + Currently infected. Order HCV genotype.

HUMAN IMMUNODEFICIENCY VIRUS (HIV) ANTIBODY TEST INTERPRETATION CLIA HIV-1/2 IgG

differentiation Interpretation

- N.D. This specimen is HIV antibody negative. A negative test does not exclude the possibility of infection with HIV. Negative results may be seen in early infection, advanced AIDS and agammaglobulinemic patients. If suspicion is high, submit a sample for HIV nucleic acid testing.

+ HIV-1 Positive for HIV-1 antibody. This sample was reactive by an HIV screening test and also reactive for HIV-1 by an HIV-1/HIV-2 IgG differentiation immunoassay. If this is the first positive result from this patient, retesting using a separately drawn sample is recommended.

+ HIV-2 Positive for HIV-2 antibody. This sample was reactive by an HIV screening test and also reactive for HIV-2 by an HIV-1/HIV-2 IgG differentiation immunoassay. If this is the first positive result from this patient, retesting using a separately drawn sample is recommended.

+ HIV HIV positive (undifferentiated). This sample was reactive by an HIV screening test and also reactive for both HIV-1 and HIV-2 by an HIV-1/HIV-2 IgG differentiation immunoassay. This sample should be considered positive for HIV antibody, however, additional tests are required to determine whether the patient is infected with HIV-1 and/or HIV-2. If this is the first positive result from this patient, retesting using a separately drawn sample is recommended.

+ Indet HIV indeterminate. HIV NUCLEIC ACID TESTING ON A REPEAT SAMPLE IS RECOMMENDED. This sample was reactive by an HIV screening test, but nonreactive by an HIV-1/HIV-2 IgG differentiation immunoassay. This could be a false positive screening test result, or an early HIV infection, and should be evaluated by HIV nucleic acid testing using a new specimen.

+ Indet HIV indeterminate. HIV NUCLEIC ACID TESTING ON A REPEAT SAMPLE IS RECOMMENDED. This sample was reactive by an HIV screening test, but indeterminate by an HIV-1/HIV-2 IgG differentiation immunoassay. This could be a false positive screening test result, or an early HIV infection, and should be evaluated by HIV nucleic acid testing using a new specimen.


18 V. SERVICES OFFERED AT THE VIROLOGY REFERENCE LABORATORY, VA-CT

Virology Reference Laboratory, VA Connecticut Health System Director: David Peaper, M.D., Ph.D, 932-5711, ext 3544

Hours of Operation: 7:30 a.m. to 4:30 p.m. Mon-Fri Location: Building 5, 2nd floor, Room C-202

Lab Phone numbers: 937-3441 (outside direct dial); or 932-5711, ext. 3379, 3380

SERVICES OFFERED Serology Assays Viral Serology

• HIV-1/2 antibody + p24 Ag (4th Generation Assay) • HIV-1/2 Differentiation Assay • Hepatitis viruses (HAV IgM, HAV IgG, HBsAg, HBsAb, HBc IgM, HBc Total, HCV) • MMRV (Measles, Mumps, Rubella, Varicella IgG) • EBV (VCA IgM, VCA IgG, EBNA IgG)

Other Serology • Celiac Antibodies (TTG IgA, TTG IgG, DPG IgG)

Molecular assays Quantitative Viral Load Assays (Roche Cobas)

• HIV-1 • HCV • HBV • CMV

Highly Multiplexed PCR Panels (BioFire FilmArray) • Respiratory Pathogen PCR panel: Influenza, RSV, Parainfluenza, Adenovirus, Rhinovirus/Enterovirus, hMPV, Coronaviruses, B. pertussis, M.

pneumoniae, and C. pneumoniae • Meningitis / Encephalitis PCR Panel: HSV-1, HSV-2, VZV, Enterovirus, Parechovirus, CMV, HHV-6, E. coli K1, H. influenzae, L. monocytogenes,

N. meningitidis, S. agalactiae, S. pneumoniae, C. neoformans/gattii Antiviral Drug Resistance

• HIV-1 Antiviral Resistance Genotyping • HCV NS5a Antiviral Resistance Sequencing

Other PCR Assays • CMV PCR (Qualitative) • HSV-1, HSV-2 and VZV Real-time PCR for detection in lesions • High Risk Human Papilloma Virus PCR from ThinPrep specimens • C. trachomatis and N. gonorrhea PCR from urine and genital specimens (Roche PCR Media)

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